Project Summary The detailed mechanisms of Human Immunodeficiency Virus Type 1 (HIV-1) assembly constitute a significant gap in our understanding of the infection cycle of this devastating pathogen. Specifically, the mechanisms by which the essential Envelope (Env) glycoprotein and structural Gag molecule efficiently coalesce to form an infectious HIV-1 particle are poorly understood, due to our inability to quantitatively measure the spatiotemporal dynamics of individual molecules at the nanoscale within infected host cells. This proposal aims to delineate the underpinnings of HIV-1 assembly on a single molecule basis and in a native cellular context. To accomplish these aims, we propose to utilize superresolution microscopy to visualize and quantify the stages of virus assembly in order to determine the role of key peptide motifs found in HIV-1 Env and Gag. Our preliminary data and analytics demonstrate our ability to quantitatively map the biogenesis of HIV-1 and strongly suggest that this approach will provide unequivocal insight in the mechanisms of particle assembly. These discoveries will provide new quantitative models to guide approaches aimed at thwarting HIV-1 assembly.